Signaling pathways underlying eosinophil cell motility revealed by using caged peptides.
Insights into structure-function relations of many proteins opens the possibility of engineering peptides to selectively interfere with a protein's activity. To facilitate the use of peptides as probes of cellular processes, we have developed caged peptides whose influence on specific proteins can be suddenly and uniformly changed by near-UV light. Two peptides are described which, on photolysis of a caging moiety, block the action of calcium-calmodulin or myosin light chain kinase (MLCK). The efficacy of theses peptides is demonstrated in vitro and in vivo by determining their effect before and after photolysis on activities of isolated enzymes and cellular functions known to depend on calcium-calmodulin and MLCK. These caged peptides each were injected into motile, polarized eosinophils, and when exposed to light promptly blocked cell locomotion in a similar manner. The results indicate that the action of calcium-calmodulin and MLCK, and by inference myosin II, are required for the ameboid locomotion of these cells. This methodology provides a powerful means for assessing the role of these and other proteins in a wide range of spatio-temporally complex functions in intact living cells.
Walker
J W
JW
Department of Physiology, University of Wisconsin, Madison, WI 53706, USA. jwwalker@facstaff.wisc.edu
Gilbert
S H
SH
Drummond
R M
RM
Yamada
M
M
Sreekumar
R
R
Carraway
R E
RE
Ikebe
M
M
Fay
F S
FS
D000818:Animals; D002024:Bufo marinus; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology*; D002465:Cell Movement; D004804:Eosinophils / Q000502:physiology*; D008845:Microinjections; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000502:physiology*; D010782:Photolysis; D012455:Salamandridae; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry
D002147:Calmodulin; D014443:Tyrosine; D009219:Myosin-Light-Chain Kinase